title: Phenobarbital-induced rat liver cytochrome P-450. Purification and characterization of two closely related isozymic forms.
abstract: The "major" phenobarbital (PB)-induced cytochrome P-450 species present in livers of male Sprague-Dawley rats was resolved into two catalytically active heme-protein fractions on diethylaminoethyl cellulose. The two species, P-450 PB-4 (Mr = 49,000) and P-450 PB-5 (Mr = 51,000), were purified to homogeneity, and their chromatographic, spectral, catalytic, and structural properties were compared. P-450 BP-5 eluted earlier on hydroxylapatite and exhibited a more significant cholate-induced Type I spectral shift than P-450 BP-4. Very similar substrate specificity profiles were evident when the two isozymes were reconstituted with lipid, cytochrome P-450 reductase, and cytochrome b5 for oxidative metabolism of several xenobiotics, although P-450 PB-4 exhibited a higher specific catalytic activity (greater than or equal to 5-fold) with all substrates tested. Marked differences were also observed in the sensitivities of both isozymes to several P-450 inhibitors. In addition, P-450 PB-4 was greater than or equal to 10-fold more susceptible than P-450 PB-5 to suicide inactivation by two allyl-containing compounds, allylisopropylacetamide and secobarbital, providing a possible explanation of the previously observed partial inactivation by such compounds of phenobarbital-induced P-450 activity in liver microsomes. One-dimensional peptide maps of the two isoenzymes were highly similar. Antibody raised against purified Long Evans rat liver P-450b (Thomas, P. E., Korzeniowski, D., Ryan, D., and Levin, W. (1979) Arch. Biochem. Biophys. 192, 524-532) cross-reacted with P-450 PB-4 and P-450 PB-5. NH2-terminal sequence analysis demonstrated that the first 31 residues of both PB-4 and PB-5 were identical. These sequences indicated that a highly hydrophobic terminal segment, observed previously for other P-450s as well, is followed by a cluster of basic residues, suggesting that the NH2-terminal portion of these P-450s might be involved in membrane anchoring. Although it is unclear whether P-450 PB-4 and P-450 PB-5 are separate gene products or are related by post-translational modifications, this present demonstration of closely related isozymic forms suggests the possible added complexity of microheterogeneity for this family of microsomal monooxygenases.
mesh_terms: D000818:Animals; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000302:isolation & purification; D007527:Isoenzymes / Q000096:biosynthesis* / Q000302:isolation & purification; D008297:Male; D008797:Metyrapone / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis*; D010634:Phenobarbital / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D013053:Spectrophotometry; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; D010634:Phenobarbital; D008797:Metyrapone